Summary/Abstract The regulation of blood vessel barrier function is one of the most important functions of the vascular network, and one of the first functions to be affected by the disease state. Despite the fact that most cardiovascular diseases affect the permeability of the blood vessel wall, our knowledge of how vascular permeability is regulated under normal conditions remains incomplete. The CCM protein complex, containing the scaffolding proteins KRIT1, CCM2, and PDCD10/CCM3, is critical for the stability of endothelial cell-cell contacts and regulation of vascular permeability in vivo. However, we still know relatively little about how these proteins are regulated in a normal functioning cell, or how this complex actually stabilizes cell-cell contacts. Preliminary data indicate that the localization of KRIT1 at endothelial adherens junctions (AJ) is essential to the proper functioning of the entire CCM complex, and that this localization is controlled by the conformation of KRIT1 and by the interaction of KRIT1 with its known binding partners. We hypothesize that the trafficking of KRIT1 to intracellular sites controls the ability of the CCM complex to regulate permeability in endothelial cells. Therefore, we propose to use several advanced imaging and biochemical approaches to determine the respective conformation and binding interactions of KRIT1 in three subcellular locations: the plasma membrane, the cytoplasm, and the nucleus. This information will be used in conjunction with functional assays to construct a model of KRIT1/CCM signaling within an intact endothelial monolayer. This information will be critical for future drug development targeting the CCM complex and will significantly increase our understanding of vascular homeostasis.